Current research in plant molecular biology is directed toward the development of improved plant varieties through the use of genetic engineering. Historically, improved plant varieties have been developed using classical genetic techniques to identify, preserve and crossbreed plants having desired traits. However, the genetic traits available to the classical breeder are limited to those that can be identified in the particular plant species the breeder is seeking to improve.
Advances in the application of the techniques of molecular biology to plants now allow for the introduction of new traits isolated from entirely different species into the plant of interest, particularly major crop plants such as cotton, maize, sorghum, soybeans, alfalfa, tobacco, and brassicas, such as rape. Traits that have been successfully transferred include insect resistance, herbicide resistance, stress tolerance, drought resistance, and disease resistance. Present day recombinant DNA technology has made it possible to identify new genes which effect the properties of plants and of products made from plants when they are transformed into new plant species. For example, a number of insect resistant varieties of cotton are presently being grown. Crop plants resistant to the herbicides Roundup®, Buctril®, and Liberty® Link are now available, as are tomatoes which can be left on the vine longer than normal tomatoes, making mechanical harvesting of tomatoes easier and cheaper.
A variety of techniques have been used to introduce foreign genes into plant cells. However, most of these techniques are limited to use with plant tissues that must be regenerated into whole plants and require a period of time in tissue culture. Methods of regenerating whole plants from cells or tissues include, micropropagation of apical and lateral meristems, organogenesis, and somatic embryogenesis. Transformation of apical meristems, lateral meristems and organogenesis produce chimeric plants, i.e., plants which have the gene encoding the newly introduced trait in only a few cells, which may or may not be in the gene in germline tissue. Plants regenerated through somatic embryogenesis are rarely chimeric. Somatic embryos are usually derived from a single cell.
One common method used to introduce foreign genes into plant cells is transformation with Agrobacterium, a relatively benign natural plant pathogen. Agrobacterium actively mediates transformation events—the integration of a gene providing a desired phenotypic trait—as part of the natural process it utilizes when it infects a plant cell. Methods for transferring foreign genes into plant cells and the subsequent expression of the inserted genes in plants regenerated from transformed cells are well known in the prior art. See for example, M. De Block et al., The EMBO Journal (1984) 3:1681; Horsch et al. Science (1985) 227:1229; and C. L. Kado (Crit. Rev: Plant. Sci. (1991) 10:1.
Certain plant species have proved to be more difficult to transform with Agrobacterium than others, particularly members of the monocotyledonous plant family. Transformation of the dicot cotton has also been particularly difficult. Since monocotyledonous plants generally do not form crown galls, it was initially assumed that the host range of Agrobacterium was restricted to dicotyledonous plants. However, Stephen L. Goldman and Anne C. F. Graves described a process for transforming plants, including the monocot corn, in U.S. Pat. Nos. 5,177,010 and 5,187,073. These methods involve making a wound in a seedling in an area containing rapidly dividing cells, then inoculating the wound with Agrobacterium. 
Roberta H. Smith and Jean H. Gould disclose a method for transforming plants via the shoot apex of a plant tissue in U.S. Pat. No. 5,164,310. These inventors teach that the method described permits rapid propagation of plants while perpetuating the unique clonal and genetic characteristics of the plant being transformed. But again, these methods have proved difficult to apply on a commercial scale due to the low numbers of transformants expressing a gene responsible for a desired trait that are actually produced.
Further improvements of methods for transforming plants by inoculating plant tissues containing rapidly dividing cells are disclosed in U.S. Pat. No. 5,169,770 issued to Paula P. Chee et al. Chee et al. demonstrate that the time after germination of infecting P. vulgaris seed with Agrobacterium-based vectors is critical to the ability of the Agrobacteria to infect meristematic cells. According to Chee et al., the amount of vascular tissue in germinating cells is rapidly increasing as differentiation proceeds. Therefore, the inoculation step must be conducted within 16 to 96 hours of germination to achieve successful transformation in this legume family of plants. Chee et al., further disclose that the transformation can be carried out with either an armed or disarmed Agrobacterium vector.
The technique known as microprojectile bombardment has been used to successfully introduce genes encoding new genetic traits into a number of crop plants, including cotton, maize, tobacco, sunflowers, soybeans and certain vegetables. See for example, U.S. Pat. No. 4,945,050, issued to Sanford; Sanford et al., Trends in Biotechnology (1988) 6:299; Sanford et al., Part. Sci. Technol. (1988) 5:27; J. J. Finer and M. D. McMullen, Plant Cell Reports (1990) 8:586–589; and Gordon-Kamm, The Plant Cell (1990) 2:603). Transformation by microprojectile bombardment is less species and genotype specific than transformation with Agrobacterium, but the frequencies of stable transformation events achieved following bombardment can be quite low, partly due to the absence of a natural mechanism for mediating the integration of a DNA molecule or gene responsible for a desired phenotypic trait into the genomic DNA of a plant. Particle gun transformation of cotton for example, has been reported to produce no more than one clonal transgenic plant per 100–500 meristems targeted for transformation. Only 0.1 to 1% of these transformants were capable of transmitting foreign DNA to progeny. See WO 92/15675. Cells treated by particle bombardment must be regenerated into whole plants, which requires labor intensive, sterile tissue culture procedures and is generally genotype dependent in most crop plants, particularly so in cotton. Similar low transformation frequencies have been reported for other plant species as well.
The methods of the prior art will provide transgenic plants in many types of plants. However, all of the presently available methods have been difficult to apply to the development of transgenic plant lives on a commercial scale due to the low numbers of transformants produced by the prior art transformation methods.
Thus, there still exists a need for procedures that will allow the delivery of a transforming agent, such as Agrobacteria carrying a foreign DNA encoding a desired trait, to germline tissues in a manner that provides efficient incorporation of the foreign DNA into the genomic DNA of the cells in these tissues. The method of the present application targets apical meristematic tissues in a manner that enhances incorporation of the foreign DNA into the genomic DNA of the plant greatly improving the frequency with which transformed plants can be produced.